Cross-linked polymers and implants derived from electrophilically activated polyoxazoline

ABSTRACT

One aspect of the invention relates to a biocompatible, covalently cross-linked, polymer that is obtained by reacting an electrophilically activated polyoxazoline (EL-POX) with a nucleophilic cross-linking agent, said electrophilically activated POX comprising m electrophilic groups; and said nucleophilic cross-linking agent comprising n nucleophilic groups, wherein the m electrophilic groups are capable of reaction with the n nucleophilic groups to form covalent bonds; wherein m≧2, n≧2 and m+n≧5; wherein at least one of the m electrophilic groups is a pendant electrophilic group and/or wherein m≧3; and wherein the EL-POX comprises an excess amount of electrophilic groups relative to the amount of nucleophilic groups contained in the nucleophilic cross-linking agent. The invention further relates to biocompatible medical products comprising such a cross-linked POX-polymer. Also provided is a kit for producing a biocompatible, cross-linked POX-polymer. The invention further provides a tissue adhesive medical product comprising at least 1% by weight of dry matter of EL-POX, said EL-POX comprising at least 2 electrophilic groups, including at least one pendant electrophilic group. The polymers according to the invention have excellent implant and/or sealing characteristics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Phase of International PatentApplication No. PCT/NL2012/050001, filed Jan. 2, 2012, published as WO2012/057628, which claims priority to European Application No.11150099.7, filed Jan. 4, 2011. The contents of these applications areherein incorporated by reference in their entirety.

TECHNICAL FIELD OF THE INVENTION

The invention relates to a biocompatible, covalently cross-linkedpolymer that is obtained by reacting electrophilically activatedpolyoxazoline (EL-POX) with a cross-linking agent.

The invention further relates to biocompatible medical productscomprising such a cross-linked POX-polymer. Examples of such medicalproducts are medical implants including bone implants, soft tissueimplants, adhesive implants, coatings on implants, sutures, adhesivetissue sealants and adhesive tissue tapes.

Also provided is a kit for producing a biocompatible, cross-linkedPOX-polymer.

The invention further provides a tissue adhesive medical productcomprising at least 1% by weight of dry matter of electrophilicallyactivated POX comprising reactive groups that are capable of reactingwith tissue components.

BACKGROUND OF THE INVENTION

Tissue adhesives have many potential medical applications, includingwound closure, supplementing or replacing sutures or staples in surgicalprocedures, adhesion of synthetic onlays or inlays to the cornea, drugdelivery devices, and as anti-adhesion barriers to prevent post-surgicaladhesions.

Conventional tissue adhesives include fibrin sealants, cyanoacrylatebased sealants, and other synthetic sealants and polymerizablemacromers. Some of these conventional sealants are only suitable for aspecific range of adhesive applications. For example,cyanoacrylate-based adhesives have been used for topical wound closure,but the release of toxic degradation products limits their use forinternal applications. Fibrin-based adhesives are expensive, often needrefrigerated storage, are slow curing, have limited mechanical strength,and pose a risk of viral infection.

For certain applications, for example, ophthalmic applications such assealing wounds resulting from trauma such as corneal lacerations, orfrom surgical procedures such as vitrectomy procedures, abdominalhernias, cataract surgery, LASIK surgery, glaucoma surgery, and cornealtransplants; neurosurgery applications, such as sealing the dura;plugging to seal a fistula or the punctum, slow degrading tissueadhesives are needed.

The last decade, several types of (semi)synthetic hydrogel tissueadhesives have been developed, which have improved adhesive propertiesand are non-toxic. Most of these hydrogel tissue adhesives, likeDuraSeal®, are chemically based on a process called PEGylation used inpolymer-modified therapeutics with polyethylene glycol (PEG) end cappedmPEG-NHS precursors like, for instance, PEG-succinimidyl glutarate.These hydrogel tissue adhesives, based on PEGylation, typically swell ordissolve away too quickly, or lack sufficient cohesion (interconnectingmechanical strength), thereby decreasing their effectiveness as surgicaladhesives. Moreover, to apply these hydrogel tissue adhesives, dualsyringe spray technology may be needed, which demands extensive samplepreparation from freeze dried starting materials. Finally, theproperties of such PEG-based materials cannot be easily controlled andthe number of NHS-groups is limited to the number of chain ends;possibly comprising multiple NHS groups per chain end resulting in ahigh NHS group density rather than regularly distributed groups.

WO 02/062276 describes a hydrogel tissue sealant comprising astar-shaped PEG-succinimidyl glutarate precursor, also known asstar-PEG-NHS or star-PEG-NS or star-SG-PEG or star-PEG-SG, that reactswith a trilysine precursor. The star-SG-PEG precursor may bereconstituted in pH 4 phosphate, while the trilysine precursor may bereconstituted in pH 8 borate buffer. Upon mixing, covalent amide bondsbetween amines of the trilysine precursor and NHS-activated terminalcarboxylate groups of the star-SG-PEG precursor are formed.

US 2003/0119985 and US 2005/0002893 describe a tissue sealant based onthe same star-PEG-NHS/trilysine principles in which hydrogels are formedby reacting a component having nucleophilic groups with a componenthaving electrophilic groups to form a cross-linked network via covalentbonding.

WO 2010/043979 describes an implant comprising: a porous layer, saidporous layer comprising a first sublayer that comprises a first hydrogelprecursor and a second sublayer free from hydrogel precursor; and afirst additional layer, said first additional layer being a non porouslayer comprising a second hydrogel precursor, wherein the first hydrogelprecursor has nucleophilic functional groups and the second hydrogelprecursor has electrophilic functional groups.

WO 02/102864 describes cross-linkable composition comprised of:

-   a) a first cross-linkable component A having m nucleophilic groups,    wherein m>2;-   b) a second cross-linkable component B having n electrophilic groups    capable of reaction with the m nucleophilic groups to form covalent    bonds, wherein n>2 and m+n>4; and-   c) a third cross-linkable component C having at least one functional    group selected from (i) nucleophilic groups capable of reacting with    the electrophilic groups of component B and (ii) electrophilic    groups capable of reacting with the nucleophilic groups of component    A, wherein the total number of functional groups on component C is    represented by p, such that m+n+p>5    wherein at least one of components A, B and C is comprised of a    hydrophilic polymer, and cross-linking of the composition results in    a biocompatible, nonimmunogenic, cross-linked matrix. Polyoxazolines    are mentioned as an example of a hydrophilic polymer.

WO 2006/078282 describes a dry powder composition comprised of: a firstcomponent having a core substituted with m nucleophilic groups, wherem>2; and a second component having a core substituted with nelectrophilic groups, where n>2 and m+n>4; wherein the nucleophilic andelectrophilic groups are non-reactive in a dry environment but arerendered reactive upon exposure to an aqueous environment such that thecomponents inter-react in the aqueous environment to form athree-dimensional composition. The core of the first or second componentcan be a hydrophilic polymer. WO 2006/078282 mentions a range ofdifferent hydrophilic polymers, including polyoxazolines.

WO 2010/033207 describes a conjugate comprising a residue of atherapeutic peptide moiety covalently attached, either directly orthrough a spacer moiety of one or more atoms, to a water-soluble,non-peptidic polymer. Polyoxazoline is mentioned as an example of awater-soluble polymer.

WO 2009/043027 describes multiarmed, monofunctional forms ofpolyoxazolines and conjugates of such polyoxazoline derivatives withdrugs. Example 18 describes the coupling of bis-amine with apolyoxazoline with the repeating unit having the structure—[N(COCH₂CH₃)CH₂CH₂]_(n)—, in which the terminal nitrogen is bound tomethyl and in which the other terminus carries the followingelectrophilic residue: —OCO₂-NHS.

U.S. Pat. No. 5,635,571 describes polyoxazolines comprising a terminalNH₂ or OH group and a pendant ester group. A hyperbranched polymer isproduced by amidation between the pendant ester group and the chainterminating NH₂ or by transesterification between the pendant estergroup and the terminating OH group.

Chujo et al. (Reversible Gelation of Polyoxazoline by Means ofDiels-Alder Reaction, Macromolecules, 1990(23), 2636-2641) describes thepreparation of a polyoxazoline hydrogel by means of intermolecularDiels-Alder reaction between furan-modified poly(N-acetylethylenimine)(PAEI) and maleimide-modified PAEI, which were synthesized from thepartially hydrolyzed PAEIs by the reaction with furan- ormaleimidecarboxylic acid, respectively, in the presence ofdicyclohexylcarbodiimide.

Luxenhofer (Thesis: Novel Functional Poly(2-oxazoline)s as PotentialCarriers for Biomedical Applications, Technische Universität München(2007)) describes poly(2-oxazoline) hydrogels. These hydrogels areprepared by crosslinking a poly(2-methyl-2-oxazoline) comprisingaldehyde sidechains with hydrazine or with a poly(2-ethyl-2-oxazoline)comprising amine sidechains. Gelation and swelling was evaluated inaqueous buffers having a pH in the range of 4 to 9.

It is of interest to expand the range of polymers having implant ortissue sealant applications, especially to provide polymers havingproperties not possessed by PEG-based polymers while being similarlybiocompatible.

SUMMARY OF THE INVENTION

The inventors have discovered that a polymer having excellent implantand/or sealing characteristics can be obtained by reacting anelectrophilically activated polyoxazoline (EL-POX) that comprises atleast two electrophilic groups with a cross-linking agent that comprisestwo or more nucleophilic groups. The EL-POX employed in the cross-linkedpolymer comprises at least 1 pendant electrophilic group or, if it doesnot, it comprises at least 3 terminal electrophilic groups.

In accordance with one embodiment of the present invention, thecross-linked polymer has tissue-adhesive properties due to the fact thatit contains unreacted electrophilic groups that are capable of reactingwith nucleophile-containing components naturally present in tissue.Thus, the invention provides a tissue-adhesive biocompatible, covalentlycross-linked, polymer that is obtained by reacting an EL-POX with anucleophilic cross-linking agent, said EL-POX comprising m electrophilicgroups; and said nucleophilic cross-linking agent comprising nnucleophilic groups, wherein the m electrophilic groups are capable ofreaction with the n nucleophilic groups to form covalent bonds; whereinm≧2, n≧2 and m+n≧5; wherein at least one of the m electrophilic groupsis a pendant electrophilic group and/or wherein m≧3; and wherein theEL-POX comprises an excess amount of electrophilic groups relative tothe amount of nucleophilic groups contained in the nucleophiliccross-linking agent.

The cross-linked polymer of the present invention provides a number ofbeneficial properties:

-   -   Adhesiveness and mechanical properties of the cross-linked        polymer can be manipulated effectively by controlling the level        and nature of alkyl side chain and/or end-group        functionalization and the polymer chain length. Cationic        2-alkyl-2-oxazoline polymerization can suitably be used to        incorporate a large number of activated groups in the alkyl side        chains of the POX polymer;    -   Cohesiveness of the cross-linked polymer is determined largely        by the number/density of cross-links within the polymer. The        number of cross-links in the polymer can be varied within wide        ranges by incorporating different amounts of activated groups in        the alkyl side chains of the POX;    -   The swelling index of the cross-linked polymer can be controlled        by manipulating the number of cross-links and the chain length        of the alkyl side chains in the POX;    -   Implants made of the cross-linked polymer are an ideal drug        depot for local drug delivery. The release of drugs, such as        antibiotics, growth factors like VEGF and osteogenic factor        (BMP-2), may be sustained by slow diffusion from the        interconnecting network depending on the nature of the alkyl        side chains and the density of cross links within the network;    -   Biodegradability of the cross-linked polymer can be controlled        effectively by incorporating hydrolysable groups, such as esters        or carbonates, into the copolymers. It is further influenced by        the number of internal cross links. Thus, it is possible to fine        tune the biodegradibility of the polymer to the intended use;    -   Based on end capped POX-NHS in research for drug delivery, POX        seems to have similar or even better stealth and antifouling        behaviour than PEG. For renal clearance the POX should have a Mw        of 30,000 or less.

Another aspect of the invention relates to a kit for producing abiocompatible, cross-linked polymer, said kit comprising the EL-POX andthe nucleophilic cross-linking agent described herein before; wherein atleast one of the m electrophilic groups is a pendant electrophilicgroup. This kit may suitably be used to deliver bone substitutematerials, anti-adhesive implants (films), adhesive implants (forinstance a tissue sealant for closing arterial puncture sites, or forembolization or to treat urinary incontinence).

The rate at which cross-linking occurs when the EL-POX and cross-linkingagent are brought together can be controlled effectively by includingnon-inert fluids, such as water (pH), alcohols and/or polyols.

The invention also provides a biocompatible medical product comprisingat least 1% by weight of dry matter of the tissue-adhesive cross-linkedpolymer of the present invention. Examples of such biocompatible medicalproducts include implants, tissue sealants, adhesive tissue tape, suturematerials, polymer coated stents and haemostatic materials.

The invention further provides a tissue adhesive medical productcomprising at least 10% by weight of dry matter of EL-POX, said EL-POXcomprising at least 2 electrophilic groups, including at least onependant electrophilic group, said reactive groups being selected fromcarboxylic acid esters, sulfonate esters, phosphonate esters,pentafluorophenyl esters, p-nitrophenyl esters, p-nitrothiophenylesters, acid halide groups, anhydrides, ketones, aldehydes, isocyanato,thioisocyanato (isothiocyanato), isocyano, epoxides, activated hydroxylgroups, olefins, glycidyl ethers, carboxyl, succinimidyl esters,succinimidyl carbonates, succinimidyl carbamates, sulfosuccinimidylesters, sulfosuccinimidyl carbonates, maleimido, ethenesulfonyl, imidoesters, aceto acetate, halo acetal, orthopyridyl disulfide,dihydroxyphenyl derivatives, vinyl acrylate, acrylamide, iodoacetamideand combinations thereof. The aforementioned reactive groups are capableof reacting with nucleophile-containing components naturally present intissue, thereby imparting tissue adhesive properties to the medicalproduct containing the EL-POX.

The tissue adhesive medical product of the present invention offers thefollowing advantages:

-   -   Due to its hydrophilic/hydrophobic balance, EL-POX is soluble in        organic fluids like ethanol and dichloromethane as well as in        water.    -   EL-POX has excellent amorphous properties with a glass        transition temperature markedly higher compared to, for        instance, polyethylene glycol.    -   EL-POX has film forming capabilities and is easy to plasticize        with limited amounts of plasticizers. An advantage of EL-POX        over electrophilically activated PEG is the possibility to        incorporate different and also functionalized groups along the        chain in order to tune the polymer properties for specific        applications;    -   POX provides a protective environment for NHS groups in the        implant when it is not exposed to water or bodily fluids.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, one aspect of the invention relates to a tissue-adhesivebiocompatible, covalently cross-linked, polymer that is obtained byreacting an electrophilically activated polyoxazoline (EL-POX) with anucleophilic cross-linking agent, said EL-POX comprising m electrophilicgroups; and said nucleophilic cross-linking agent comprising nnucleophilic groups, wherein the m electrophilic groups are capable ofreaction with the n nucleophilic groups to form covalent bonds; whereinm≧2, n≧2 and m+n≧5; wherein at least one of the m electrophilic groupsis a pendant electrophilic group and/or wherein m≧3; and wherein theEL-POX comprises an excess amount of electrophilic groups relative tothe amount of nucleophilic groups contained in the nucleophiliccross-linking agent.

The term “polyoxazoline” as used herein refers to apoly(N-acylalkylenimine) or a poly(aroylalkylenimine) and is furtherreferred to as POX. An example of POX is poly(2-ethyl-2-oxazoline). Theterm “polyoxazoline” also encompasses POX copolymers.

The terminology “pendant electrophilic group” refers to an electrophilicgroup that is comprised in a side chain, e.g. an alkyl or aryl sidechain, of the POX polymer, as opposed to an electrophilic group that islocated at a terminus of the POX polymer chain. It should be understoodthat a particular side chain of the POX polymer may suitably containmore than one electrophilic group, in which case each electrophilicgroup within that particular side chain counts as a pendantelectrophilic group.

The term “amine groups” as used herein refers to primary or secondaryamine groups.

Whenever the cross-linked polymer of the present invention ischaracterized on the basis of the presence of a certain number ofparticular groups or bonds per 100 monomers it should be understood thatthis does not imply that the polymer contains at least 100 monomers. Forexample, a cross-linked polymer comprising 80 monomers and 8 pendantgroups contains 10 pendant groups per 100 monomers. Likewise, if apolymer contains 80 monomers and it is specified that this polymercontains, for instance, at least x pendant groups per 100 monomers, thiscriterion is met if this particular polymer contains on average at least0.8× pendant groups.

Preferably, the EL-POX of the present invention is an electrophilicallyactivated version of a polyoxazoline polymer whose repeating units arerepresented by the following formula (I):(CHR¹)_(m)NCOR²R², and each of R¹ independently being selected from H, optionallysubstituted C₁₋₂₂ alkyl, optionally substituted cycloalkyl, optionallysubstituted aralkyl, optionally substituted aryl; and m being 2 or 3.The invention also encompasses the use of polyoxazolines copolymers thatcomprise two or more different repeating units that are represented byformula (I).

Preferably, R¹ and R² in formula (I) are selected from H and C₁₋₂₂alkyl, even more preferably from H and C₁₋₄ alkyl. R¹ most preferably isH. The integer m in formula (I) is preferably equal to 2.

According to a preferred embodiment, the polyoxazoline employed inaccordance with the present invention is a polymer, even more preferablya homopolymer of 2-alkyl-2-oxazoline, said 2-alkyl-2-oxazoline beingselected from 2-methyl-2-oxazoline, 2-ethyl-2-oxazoline,2-propyl-2-oxazoline, 2-butyl-2-oxazoline and combinations thereof. Mostpreferably, the polyoxazoline is a homopolymer of 2-ethyl-oxazoline.

Preferably, the electrophylically active POX (EL-POX) and thecross-linking agent employed in the present cross-linked polymer are notidentical.

The n nucleophilic groups of the nucleophilic cross-linking agent arepreferably selected from amine groups, thiol groups and combinationsthereof. Most preferably, these nucleophilic groups are amine groups.

EL-POX can be activated in its side chains, at its termini (provided itcontains at least 3 terminal elecrophilic groups), or both. An exampleof a terminal, end capped, EL-POX is a succinimidyl succinate ester likeCH₃O-POX-O₂C—CH₂—C(CH₂CO₂₋NHS)₃. An example of a side chain activatedEL-POX is POX containing NHS groups in the alkyl side chain. Yet anotherexample of EL-POX are star-shaped polymers end-functionalized with anNHS-esters.

The electrophilic groups contained in the EL-POX preferably are highlyreactive towards amine groups or thiol groups, preferably at ambientand/or physiological conditions. Thus, unlike U.S. Pat. No. 5,635,571,the electrophilic groups preferably are not pendant ester groups havingthe following formula —(CO)R(CO)OR², wherein R is phenylene or alkylenecontaining 2 to 18 carbon atoms, and R² is C₁₋₄ alkyl.

The electrophilic groups present in the EL-POX are preferably selectedfrom: carboxylic acid esters, sulfonate esters, phosphonate esters,pentafluorophenyl esters, p-nitrophenyl esters, p-nitrothiophenylesters, acid halide groups, anhydrides, ketones, aldehydes, isocyanato,thioisocyanato (isothiocyanato), isocyano, epoxides, activated hydroxylgroups, olefins, glycidyl ethers, carboxyl, succinimidyl esters,succinimidyl carbonates, succinimidyl carbamates, sulfosuccinimidylesters, sulfosuccinimidyl carbonates, maleimido (maleimidyl),ethenesulfonyl, imido esters, aceto acetate, halo acetal, orthopyridyldisulfide, dihydroxy-phenyl derivatives, vinyl, acrylate, acrylamide,iodoacetamide and combinations thereof.

More preferably, the electrophilic groups present in the EL-POX areselected from: carboxylic acid esters, acid chloride groups, anhydrides,ketones, aldehydes, isocyanato, thioisocyanato, epoxides, activatedhydroxyl groups, olefins, carboxyl, succinimidyl ester, succinimidylcarbonate, succinimidyl carbamates, sulfosuccinimidyl ester,sulfosuccinimidyl carbonate, maleimido, ethenesulfonyl and combinationsthereof. Even more preferably, the electrophilic groups present in theEL-POX are selected from aldehydes, isocyanato, thioisocyanato,succinimidyl ester, sulfosuccinimidyl ester, maleimido and combinationsthereof

Most preferably, the electrophilic groups present in the EL-POX areselected from isocyanato, thioisocyanato, succinimidyl ester,sulfosuccinimidyl ester, maleimido and combinations thereof.

Examples of sulfonate esters that can be used as electrophilic groupsinclude mesylate, tosylate, nosylate, triflate and combinations thereof.Examples of olefins that can be employed include acrylate, methacrylate,ethylacrylate and combinations thereof. Examples of activated hydroxylgroups include hydroxyl groups that have been activated with anactivating agent selected from p-nitrophenyl chlorocarbonates,carbonyldiimidazoles (e.g. 1,1-carbonyl diimidazole) and sulfonylchloride.

According to one preferred embodiment, the nucleophilic groups of thenucleophilic cross-linking agent are amine groups and the electrophilicgroups comprised in the EL-POX are selected from carboxylic acid esters,sulfonate esters, phosphonate esters, pentafluorophenyl esters,p-nitrophenyl esters, p-nitrothiophenyl esters, acid halide groups,anhydrides, ketones, aldehydes, isocyanato, thioisocyanato, isocyano,epoxides, activated hydroxyl groups, glycidyl ethers, carboxyl,succinimidyl esters, succinimidyl carbonates, succinimidyl carbamates,sulfosuccinimidyl esters, sulfosuccinimidyl carbonates, imido esters,dihydroxy-phenyl derivatives, and combinations thereof.

Examples of succinimidyl derivatives that may be employed includesuccinimidyl glutarate, succinimidyl propionate, succinimidylsuccinamide, succinimidyl carbonate, disuccinimidyl suberate,bis(sulfosuccinimidyl) suberate, dithiobis(succinimidylpropionate),bis(2-succinimidooxycarbonyloxy) ethyl sulfone and3,3′-dithiobis(sulfosuccinimidyl-propionate). Examples ofsulfosuccinimidyl derivatives that can be used includesulfosuccinimidyl(4-iodoacetyl)aminobenzoate, bis(sulfosuccinimidyl)suberate,sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate,dithiobis-sulfosuccinimidyl propionate, disulfo-succinimidyl tartarate;bis[2-(sulfo-succinimidyloxycarbonyloxyethylsulfone)], ethylene glycolbis(sulfosuccinimiclylsuccinate), dithiobis-(succinimidyl propionate).Examples of dihydroxyphenyl derivatives include dihydroxyphenylalanine,3,4-dihydroxyphenylalanine (DOPA), dopamine, 3,4-dihydroxyhydroccinamicacid (DOHA), norepinephrine, epinephrine and catechol.

Even more preferably, the electrophilic groups contained in the EL-POXare selected from aldehydes, succinimidyl esters, succinimidylcarbonates, succinimidyl carbamates, imido esters, dihydroxyphenylderivatives and combinations thereof. Most preferably, the electrophilicgroups are selected from the group of N-hydroxysuccinimide esters,aldehydes, dihydroxyphenyl derivatives and combinations thereof.

According to another preferred embodiment, the nucleophilic groups ofthe nucleophilic cross-linking agent are thiol groups and theelectrophilic groups contained in the EL-POX are selected from haloacetals, orthopyridyl disulfide, maleimides, vinyl sulfone,dihydroxyphenyl derivatives, vinyl, acrylate, acrylamide, iodoacetamide,succinimidyl ester, succinimidyl carbonate, succinimidyl carbamates,sulfosuccinimidyl ester, sulfosuccinimidyl carbonate and combinationsthereof. More preferably, the electrophilic groups are selected fromsuccinimidyl esters, halo acetals, maleimides, or dihydroxyphenylderivatives and combinations thereof. Most preferably, electrophilicgroups are selected from maleimides or dihydroxyphenyl derivatives andcombinations thereof.

The benefits of the present invention are particularly appreciated incase the number of electrophilic groups m that is contained in theEL-POX is at least 13.

The EL-POX according to the present invention is advantageously derivedfrom a copolymer of 2-NHS-functional-2-oxazoline and2-alkyl-2-oxazoline. The 2-NHS-functional-2-oxazoline comprised in thecopolymer is preferably selected from mercaptoalkanoate NHS-esterfunctionalized 2-alkenyl-2-oxazoline or 2-NHS alkanoate-2-oxazoline andcombinations thereof. Even more preferably, the2-NHS-functional-2-oxazoline is selected from mercaptoacetic acidNHS-ester functionalized 2-butenyl-2-oxazoline, 2-NHSpropionate-2-oxazoline and combinations thereof.

The NHS-ester may already be present in monomers employed during polymersynthesis or it may be introduced afterwards by coupling to a functionalcomonomer or by partial hydrolysis of POX followed by alkylation oramidation of the resulting secondary amine groups in the polymer chain.The 2-alkyl-2-oxazoline is preferably selected from 2-ethyl-2-oxazoline,2-methyl-2-oxazoline, 2-propyl-2-oxazoline and combinations thereof.Typically, the EL-POX contains 2-NHS-functional-2-oxazoline and2-alkyl-2-oxazoline in a molar ratio that lies within the range of 1:33to 1:2, more preferably of 1:20 to 1:5.

In accordance with another advantageous embodiment the EL-POX accordingto the present invention is a copolymer of 2-alkyl-2-oxazoline and2-maleimido-functionalized-2-oxazoline. The latter maleimido unit can beintroduced by post-modification of amine or acid side chainfunctionalities or of backbone secondary amines resulting from partialhydrolysis.

The EL-POX of the present invention advantageously contains one or morependant electrophilic groups. Typically, the EL-POX contains 3 to 50pendant electrophilic groups per 100 monomers, more preferably 4 to 35pendant electrophilic groups per 100 monomers, even more preferably atleast 5 to 20 pendant electrophilic groups per 100 monomers.

As explained herein before, more than one electrophilic group may becontained in a side chain of the EL-POX. Preferably, however, thependant electrophilic groups of the EL-POX are located in different sidechains.

The EL-POX employed in accordance with the present invention typicallyhas a molecular weight in the range of 1,000 to 100,000 g/mol, morepreferably of 5,000 to 50,000 and most preferably of 10,000 to 30,000g/mol.

According to a particularly preferred embodiment, the n nucleophilicgroups present in the nucleophilic cross-linking agent are primary aminegroups.

In one embodiment of the invention the nucleophilic cross-linking agentemployed in the cross-linked polymer is a low molecular weight polyaminehaving a molecular weight of less than 1,000 g/mol, more preferably ofless than 700 g/mol and most preferably of less than 400 g/mol. Evenmore preferably, the nucleophilic cross-linking agent is selected fromthe group of dilysine; trilysine; tetralysine; pentalysine; dicysteine;tricysteine; tetracysteine; pentacystein; oligopeptides comprising twoor more amino acid residues selected from lysine, ornithine, cysteine,arginine and combinations thereof, and other amino acid residues;spermine; tris(aminomethyl)amine; arginine and combinations thereof.

According to another embodiment of the invention, the nucleophiliccross-linking agent employed in the cross-linked polymer is a highmolecular weight polyamine selected from the group of: nucleophilicallyactivated POX (NU-POX) comprising at least two amine groups; chitosan;chitosan derivatives (e.g. dicarboxy-derivatised chitosan polymers asdescribed in WO 2009/028965), polyethyleneimines; polyvinylamine;polyallyl amine; amine-functionalized poly(meth)acrylates;polysaccharides containing amine-functional moieties such aspoly(galactosamine); styrenics; polypeptides comprising two or moreamino acid residues selected from lysine, ornithine, cysteine, arginineand combinations thereof, and other amino acid residues; andcombinations thereof. Albumin of natural source or recombinant is anexample of a polypeptide that may suitably be employed as a polypeptide.Amine-functionalized polyethylene glycol is another example of a highmolecular weight polyamine that can suitably be employed as thenucleophilic cross-linking agent.

The high molecular weight cross-linking agent typically has a molecularweight of at least 2,000 g/mol, even more preferably of at least 10,000g/mol.

Preferably, the nucleophilic cross-linking agent is not apharmaceutically active peptide comprising more than five, morepreferably more than two amino acid residues (as opposed to theconjugates described in WO 2010/033207). Most preferably, thecross-linking agent is not a pharmaceutically active peptide. Here theterm “pharmaceutically active peptide” means that the pharmaceuticallyactivity of the peptide has been demonstrated scientifically.

According to a particularly preferred embodiment the high molecularweight polyamine is NU-POX comprising at least two amine groups. Evenmore preferably, the high molecular polyamine is derived from ahomopolymers or copolymer of cysteamine modified 2-alkenyl-2-oxazolineor 2-t-BOC-aminoalkyl-2-oxazoline and 2-alkyl-2-oxazoline. Thecysteamine modified 2-alkenyl-2-oxazoline comprised in the copolymer ispreferably selected from 2-butenyl-2-oxazoline, and combinationsthereof. The 2-alkyl-2-oxazoline is preferably selected from2-ethyl-2-oxazoline, 2-methyl-2-oxazoline, 2-propyl-2-oxazoline andcombinations thereof. The amine moieties can also be introduced bypartial hydrolysis of POX followed by alkylation or amidation of theresulting secondary amine groups in the polymer chain. Typically, theNU-POX contains cysteamine modified 2-alkenyl-2-oxazoline and2-alkyl-2-oxazoline in a molar ratio that lies within the range of 1:33to 1:2, more preferably of 1:20 to 1:5.

According to another preferred embodiment, the n nucleophilic groupspresent in the nucleophilic cross-linking agent are thiol (sulfohydryl)groups.

In one embodiment of the invention the nucleophilic cross-linking agentemployed in the cross-linked polymer is a low molecular weight polythiolcomprising 2 or more thiol groups having a molecular weight of less than1,000 g/mol, more preferably of less than 700 g/mol and most preferablyof less than 400 g/mol. Even more preferably, the nucleophiliccross-linking agent is selected from the group of trimercaptopropane,ethanedithiol, propanedithiol, 2-mercaptoethyl ether,2,2′-(ethylenedioxy)diethanethiol, tetra(ethylene glycol) dithiol,penta(ethylene glycol) dithiol, hexaethylene glycol dithiol; thiolmodified pentaerythritol, dipentaerythritol, trimethylolpropane orditrimethylolpropane; oligopeptides containing at least two cysteineunits.

According to another embodiment of the invention, the nucleophiliccross-linking agent employed in the cross-linked polymer is a highmolecular weight polythiol selected from the group of: NU-POX comprisingat least two thiol groups; thiol-functionalized poly(meth)acrylates;polysaccharides containing thiol-functional moieties; styrenics;polypeptides comprising two or more thiol groups.

According to a particularly preferred embodiment the high molecularweight polythiol is NU-POX comprising at least two thiol groups. Evenmore preferably, the high molecular polythiol is apoly(2-alkyl-2-oxazoline) with thiol terminal groups that have beenintroduced by endcapping the polymerization with a multifunctionalinitiator with potassium xanthogenate followed by aminolysis to get thefree thiol groups. Alternatively, the thiol moieties are introduced intothe side chains of the NU-POX by copolymerization of a protected thiolcontaining monomer or by modification of acid, amine or alkenyl sidechains or by modification of the backbone secondary amines resultingfrom partial hydrolysis. The 2-alkyl-2-oxazoline is preferably selectedfrom 2-ethyl-2-oxazoline, 2-methyl-2-oxazoline, 2-propyl-2-oxazoline andcombinations thereof.

The NU-POX typically has a molecular weight in the range of 1,000 to100,000 g/mol, more preferably of 10,000 to 30,000 g/mol.

According to a particularly preferred embodiment the m electrophilicgroups comprised in the EL-POX component of the cross-linked polymer arecapable of reacting with the n nucleophilic groups of the cross-linkingagent under ambient and/or physiological conditions to form covalentbonds. Most preferably, the electrophilic groups are capable of reactingwith the nucleophilic groups to form covalent bonds under ambientcondition. The ability to form cross-links at ambient temperature isespecially advantageous if the cross-linking should occur in situ duringe.g. surgery. Typically, at 35° C. and 1 atm., the cross-linkingreaction between the EL-POX and the cross-linking agent is completedwithin 30 minutes, preferably within 10 minutes, more preferably within5 minutes, most preferably within 2 minutes.

As explained herein before, for certain applications it can beadvantageous if the cross-linked polymer contains free electrophilicgroups that have not reacted with the nucleophilic groups of thenucleophilic cross-linking agent. Such a cross-linked polymer isobtained by employing EL-POX comprising an excess amount ofelectrophilic groups relative to the amount of nucleophilic groupscontained in the cross-linking agent. Thus, in a preferred embodiment,the ratio m/n exceeds 1.2, more preferably exceed 1.5, most preferablyexceeds 2.0. Typically, the latter ratio does not exceed 1000, morepreferably it does not exceed 200, even more preferably it does notexceed 50 and most preferably it does not exceed 10.

The non-reacted electrophilic groups in the cross-linked polymer of thepresent invention impart tissue adhesive properties to the polymer asthey can react with nucleophilic groups (e.g amino groups and thiolgroups) that are naturally present in tissue. Thus, when a cross-linkedpolymer containing non-reacted electrophilic groups is applied totissue, the non-reacted electrophilic groups can react with nucleophilicgroups of the tissue, thereby creating strong adhesion between thepolymer and the tissue.

The tissue adhesive properties of the cross-linked polymer can becontrolled very effectively by manipulating the number of non-reactedelectrophilic groups in the cross-linked polymer. Generally speaking,the higher the number of non-reacted electrophilic groups, the strongerthe adhesion.

Thus, in accordance with a particularly preferred embodiment of theinvention the cross-linked polymer comprises, per 100 monomers, at least1, more preferably in between 2 and 95, even more preferably in between5 and 70, most preferably in between 10 and 50 electrophilic groups thatare contained in the EL-POX component of the cross-linked polymer andthat have not reacted with a nucleophilic group of the nucleophiliccross-linking agent.

Of the total number of reacted and non-reacted elecrophilic groups inthe cross-linked polymer the non-reacted electrophilic groups preferablyrepresent 10-95%, more preferably 20-80% and most preferably 40-60%.

The cross-linked polymer is typically obtained by reacting the EL-POXwith the nucleophilic cross-linking agent in a molar ratio of 50:1 to1:1, more preferably in the range of 10:1 to 3:2

Advantageously, 3-50%, more preferably 4-35% and most preferably 5-20%of the side chains of the EL-POX contain an electrophilic group. Asexplained herein before, cross-linked polymers obtained by cross-linkingan EL-POX having a number of pendant electrophilic groups, as opposed tocross-linked polymer that are obtained by cross-linking an EL-POX withonly terminal electrophilic groups, offer a number of advantages,including the possibility to tune characteristics such as cohesion,mechanical strength, swellabilty, biodegradability and a stickinessacross much wider ranges than is possible with polymers that have beenend-capped with electrophilic groups. In this respect is noted thatelectrophilically activated PEG, which is described in a large number ofprior art publications related to medical implants, does not contain anyside chains that enable the incorporation of pendant elecrophilicgroups.

The ability of the (dry) cross-linked polymer to swell when contactedwith water depends strongly on the level of cross-linking. The morecross linking, the lower the swelling index. Preferably, the polymercontains, per 100 monomers, not more than 50, more preferably not morethan 20 and most preferably not more than 10 of covalent bonds that havebeen formed by the reaction between the electrophilic groups of theEL-POX and the nucleophilic groups of the cross-linking agent.Typically, this number of covalent of bonds is at least 2 per 100monomers.

The EL-POX employed in the present cross-linked polymer typically has amolecular weight of at least 1,000 g/mol, more preferably of5,000-100,000 g/mol and most preferably of 10,000-30,000 g/mol.

EL-POX comprising pendant electrophilic groups and NU-POX comprisingpendant nucleophilic groups can suitably be prepared by cationicpolymerization of the 2-oxazoline monomers initiated by variouselectrophilic species, for example alkyl halides, sulfonic esters,strong acids and others. POX-derivatives containing activated groups inthe side chain can be prepared directly from the 2-oxazoline monomercontaining the required group, or by a polymer analogous reaction ofpolymer precursors. Preparation of POX containing hydroxyl, amino,carboxyl, mercapto, and aldehyde groups in the side chain has beendescribed in literature.

Synthesis of a functional group at the POX terminus is, for instance,described by Anna Mero et. al. (Synthesis and characterization ofpoly(2-ethyl 2-oxazoline)-conjugates with proteins and drugs: Suitablealternatives to PEG-conjugates?, Journal of Controlled Release 125(2008) 87-95).

The EL-POX employed in accordance with the present invention can be ahomopolymer or a copolymer. Most preferably, EL-POX is a copolymer.

An EL-POX copolymer, notably an amphiphilic copolymer, can be preparedin several different ways. It is feasible, for instance, to polymerize2-oxazolines containing an (protected) electrophilic or nucleophilicgroup with a hydrophilic or lipophilic comonomer. It is also possible topartially hydrolyze POX followed by modification of the resultingsecondary amine groups in the polymer. Another type of polymer can beprepared by block copolymerizations of activated 2-alkyl-2-oxazolineswith other polymers such as polylactides, polycaprolactone orpolyethyleneglycol.

Copolymers of EL-POX containing ethyl and NHS groups in the alkyl sidechain can be synthesized by cationic polymerization of2-ethyl-2-oxazoline (ETOX) and 2-(3-butenyl)-2-oxazoline (BUTOX) toyield a poly[2-(ethyl/3-butenyl)]-2-oxazoline copolymer. Thispoly[2-(ethyl/3-butenyl)]-2-oxazoline copolymer can be functionalizedwith 3-mercaptopropionic-N-hydroxysuccinimide ester or in a two-stepapproach by first thiol-ene coupling of mercaptopropionic acid followedby activation with NHS to synthesize NHS-side chain activatedpoly[2-(ethyl/3-butenyl)]-2-oxazoline copolymer.

NU-POX containing ethyl and amino (—NH₂) groups in the alkyl side chaincan be synthesized in a similar fashion by reaction ofpoly[2-(ethyl/3-butenyl)]-2-oxazoline copolymer with cysteamine or bypartial hydrolysis of POX followed by post-modification.

NU-POX containing ethyl and thiol (—SH) groups in the alkyl side chaincan be prepared in a similar fashion by reaction ofpoly[2-(ethyl/3-butenyl)]-2-oxazoline copolymer with an excess ofethanedisulfide or by copolymerization of 2-ethyl-2-oxazoline with amonomer bearing a protected thiol group.

In case the present cross-linked polymer is obtained by cross-linking anNHS-activated EL-POX, biodegradability can range from essentiallynon-degradable to easily degradable. Biodegradability can be improved byincorporation of an ester link that is more readily hydrolyzed than thesecondary amides resulting from reactions between the NHS-ester andamines. The esters are slowly hydrolyzed in aqueous environment whilethe secondary amides are mainly hydrolyzed by enzymatic degradation thatwill be very slow in the cross-linked network.

Cationic polymerization enables the synthesis of copolymers with apredefined number of activated groups. Furthermore, cationic2-alkyl-2-oxazoline polymerization can be used to incorporate a largenumber of active groups as these active groups are coupled to thenumerous alkyl side chains of the POX. This again makes it possible toproduce highly cross-linked polymers with excellent cohesive propertiesas well as outstanding adhesive properties in case the cross-linkedpolymer has a high density of unreacted electrophilic groups.

In addition, cationic polymerization allows the incorporation of variousfunctional groups in the side-chain and/o terminus, thereby enhancingthe versatility of the POX polymer system.

The synthesis of a copolymer containing a predefined number of activatedgroups per copolymer can be carried out as follows: a copolymer issynthesized by cationic polymerization of ETOX and BUTOX, as describedpreviously, to yield a poly[2-(ethyl/3-butenyl)]-2-oxazoline copolymer.Upon functionalization with 3-mercaptopropionic-N-hydroxysuccinimideester (either directly or in two steps by first introducingmercaptopropionic acid followed by NHS activation) via radical thiol-enecoupling, NHS activated side chains are introduced. In case the initialETOX/BUTOX molar ratio is 90/10, the percentage of reactive groups perpoly[2-(ethyl/3-butenyl)]-2-oxazoline copolymer, will be 10%. Increasingthe BUTOX in the initial 90/10 ETOX/BUTOX mixture will eventually resultin a higher percentage of —NHS groups, i.e. more than 10%. The exactnumber of incorporated NHS groups can be controlled by the ratio ofmonomer to initiator that determines the length of the polymer. Forexample, using an ETOX/BUTOX ratio of 90/10 and a monomer to initiatorratio of 100:1 yields polymers with 100 repeat units comprising 10 NHSgroups. When the monomer to initiator ratio is changed to 200:1 theresulting copolymer will have 20 NHS groups with the same ETOX/BUTOXratio. A similar procedure can be followed for the synthesis ofPOX-maleimide, whereby a thiol-modified maleimide (furan protected) isadded to the poly[2-(ethyl/3-butenyl)]-2-oxazoline copolymer by radicalthiol-ene coupling. Deprotection of the furan-maleimide yields thePOX-maleimide as EL-POX.

The cross-linked polymer of the present invention may be biodegradableor non-biodegradable. Preferably, the polymer is biodegradable.

A further aspect of the invention relates to a biocompatible medicalproduct comprising at least 1%, preferably at least 25% and mostpreferably 50-100% by weight of dry matter of a tissue-adhesivecross-linked polymer as defined herein before. Examples of such medicalproducts include implants, tissue sealants, adhesive tissue tape,adhesive tissue film, suture material, polymer coated stents andhaemostatic (porous) materials.

EL-POX, the cross-linking agent and combinations of EL-POX and thecross-linking agent can be formed into shape by solvent casting or hotmelt extrusion leading to, for instance, an amorphous film or tape. Thereaction between electrophilic groups of the EL-POX and the nucleophilicgroups of the crosslinking agent results in cross-linking and the excessof electrophilic groups enables the cross-linked polymer to form linksto tissue. Thus, the present invention enables the preparation ofmedical products that combine cohesiveness with adhesiveness.

A very important property of POX films is that upon thorough drying theyprotect the interior from hydrolysis. Thus, excess (non-reacted)electrophilic groups in the EL-POX or in the crosslinked polymer willretain their activity upon storage. When a medical product containingnon-reacted electrophilic groups is applied to tissue anelectrophilic-nucleophilic reaction will occur between theseelectrophilic groups and nucleophilic reactants, notably amino or thiolgroups present in tissue, thereby forming a cross linked externalnetwork (adhesion) via covalent bonding. Accordingly, the medicalproduct advantageously contains less than 10%, even more preferably lessthan 5% and most preferably less than 1% water by weight of thecross-linked polymer.

The medical product preferably also contains not more than a limitedamount of organic solvent. Preferably, the product contains less than5%, more preferably less than 0.5% organic solvent by weight of thecross-linked polymer.

For e.g. a tissue sealing tape, EL-POX and cross-linking agent can bemixed in any ratio needed by solvent casting or hot melt extrusion. Thisapproach enables very precise fine tuning of the properties needed foran adhesive tissue tape. In case the amount of electrophilic groups inthe EL-POX is high and the number of nucleophilic groups provided by thecross-linking agent is relatively low, cohesion of the resultingcross-linked polymer will be low and adhesion to tissue will be high.Naturally, it is also feasible to produce an adhesive tissue tape withhigh cohesion and high adhesion by combining a relatively high amount ofcross-linking agent with an excess amount of electrophilic groups in theEL-POX.

A particularly advantageous embodiment of the present medical product isan adhesive tissue tape or an adhesive tissue film. The presentinvention enables the preparation of a tape or film with excellenttissue-adhesive properties due to the presence of non-reactedelectrophilic groups that are capable of reacting with nucleophilicgroups naturally present in tissue. Furthermore, the cross-linkedpolymer of the present invention, due to its amorphous properties andtunable glass transition temperature, enables the preparation ofcohesive, flexible and resilient tapes and films as well as tough andharder materials tailor-made for specific applications.

As explained herein before it is highly advantageous if the cross-linkedpolymer contained in the medical product is in an amorphous state. Theterm “amorphous” refers to a material that is a solid and in which thereis no long-range order of the positions of the molecules. This lack oforder distinguishes amorphous solids from crystalline solids.

It is further preferred that the cross-linked polymer has a glasstransition temperature of at least −50° C., more preferably at least 0°C. and most preferably at least 20° C. Typically, the glass transitiontemperature of the polymer does not exceed 120° C.

The adhesive tissue tape of adhesive tissue film of the presentinvention offers the advantage that it can absorb substantial quantitiesof water to form a cohesive hydrogel. The absorption of water may resultin significant swelling of the product. However, it is also possible todesign the product in such a way that it does not swell significantlywhen it is brought into contact with moisture.

In accordance with one advantageous embodiment, the medical product doesnot exhibit significant swelling when it comes into contact withmoisture. Typically, such a medical product has a swelling index of notmore than 100%, preferably of not more than 30% and most preferably ofnot more than 10%. Medical products having a low swelling indcx cansuitably be applied in repair of dural defects and spinal cord repair

In accordance with another preferred embodiment, the medical productshows significant swelling when it is allowed to absorb water.Typically, such a swellable product has a swelling index of 100-1000%. Amedical product having a high swelling index may suitably be used tostem blood, e.g. during or after surgery.

The swelling index is determined by recording the exact weight of asample of about 2 g of fully swollen material. Next, the swollenmaterials is immersed into 20 mL of PBS buffer and incubated for 24hours at 37° C. in an air forced oven. Next, the material is dried withpaper towel and weighed. Swelling is calculated based on the followingformula:Swelling Index %=(W _(t)−W _(o))/W _(o)×100where:

W_(o) is the pre-incubation weight of the swollen material; and

W_(t) is the post-incubation weight of the swollen material.

Greater cross-linking creates a tighter network, which will decreaseswelling. This can be advantageous in certain applications, for example,if the medical product is an implant, a suture material or a tissuesealant that is applied in tight locations where gel swelling canpotentially cause adverse effects. In that case, the swelling indexpreferably does not exceed 50%, most preferably it does not exceed 10%.

The present invention enables the preparation of an adhesive tissue tapeor tissue film having excellent properties without requiring, forinstance, support layers. Accordingly, the adhesive tissue tapepreferably is a single layer tape. Likewise, the adhesive tissue filmpreferably is a single layer film.

In accordance with another advantageous embodiment, the present medicalproduct is an implant. According to a particularly preferred embodiment,the crosslinked polymer contained in the implant provides a matrix tosupport tissue regeneration. Preferably, this is a biodegradablethree-dimensional bioresorbable porous construct with attachingproperties to bone material and appropriate mechanical properties toguide cellular attachment and subsequent tissue formation. For bonereconstruction, the construct is preferably also load bearing, meaningthat any fluid component in the implant, or the in-situ formed implant,should be kept as low as possible. In this respect it is advantageousthat only very limited amounts of plasticizers like triacetin or waterare needed to render the EL-POX and NU-POX extrudable.

According to a particularly preferred embodiment, the polymer networkcomprised in the present implant contains osteoconductive fillers likebone graft materials, including autologous bone, autologous boneparticulate, allogenic bone graft material, human cadaver bone,xenograft bone graft material, animal bone, growth factors or syntheticmaterials such as hydroxyapatite, tricalcium phosphate and bioactiveglass.

Another aspect of the invention relates to a kit for producing abiocompatible, cross-linked polymer of the present invention, said kitcomprising the EL-POX and the nucleophilic cross-linking agent asdefined herein before. Preferably, at least one of the m electrophilicgroups of the EL-POX is a pendant electrophilic group.

Independently, the EL-POX and the nucleophilic cross-linking agent maybe provided in the form of fluid or a powder. If provided in powderform, the powder should be readily dispersible in a fluid which may becontained within the same kit.

The cross-linked polymer that can be produced with the present kit mayor may not have tissue-adhesive properties. For some applications it isbeneficial if the kit can be used to produce a cross-linked polymer withtissue-adhesive properties. Thus, advantageously, the EL-POX containedin the present kit comprises an excess amount of electrophilic groupsrelative to the amount of nucleophilic groups contained in thenucleophilic cross-linking agent. Thus, the EL-POX and the nucleophiliccross-linking agent can react to form a tissue-adhesive cross-linkedpolymer as defined herein before.

In a preferred embodiment, the kit comprises separately packaged volumesof the EL-POX and the nucleophilic agent. In order to allow the EL-POXand the cross-linking agent to undergo a rapid cross-linking reaction,it is preferred to provide both the EL-POX and the cross-linking agentin the form of a fluid. More specifically, it is preferred that theEL-POX is contained in a first solvent in a concentration of 1-95 wt. %and that the nucleophilic cross-linking agent is contained in a secondsolvent in a concentration of 1-95 wt. %.

According to a particularly preferred embodiment, the first solvent andthe second solvent are selected from water, polyols, alcohols (e.g.ethanol or iso-propanol) and combinations thereof. The volume containingEL-POX and/or the volume containing the cross-linking agentadvantageously contains polyol, water or a mixture of both. Furthermore,the latter volumes may suitably be buffered. The solvent(s) andbuffering system employed in the present kit are suitable chosen so asto achieve an optimum cross-linking rate.

Polyols that can suitably be employed in the present kit includeglycerol, diacetin, triacetin, sorbitol and combinations thereof.

According to a particularly preferred embodiment the first solvent andthe second solvent contain 5-50 wt. % water, more preferably 10-30 wt. %water and most preferably 15-20 wt. % water.

In-situ cross-linking characteristics can be improved by plasticizingEL-POX, and optionally NU-POX, with a plasticizer selected from thegroup of triacetin, glycerol, triethylamine and combinations thereof.Typically, the plasticizer is employed in a concentration of 1-50%, morepreferably 3-15% by weight of the EL-POX and/or NU-POX.

The separately packaged volumes of the EL-POX and the nucleophilic agentmay suitably contain a pH modifier. An acidulant is advantageouslyemployed in accordance with the present invention to shift themicro-environmental pH of the EL-POX containing volume or the crosslinked EL-POX-formulations to more acidic conditions in the presence ofwater or bodily fluids. The term “micro-environmental pH” refers to thepH within and in the vicinity of the EL-POX of the cross linkedEL-POX-formulations.

Examples of acidulants that may suitably be employed include organic andinorganic components that are capable of releasing protons, for examplean organic or inorganic acid, an acidic polymer, e.g. a carbomer, or alatent acid (e.g. glucono-δ-lactone). Preferably, the acidulant has apK_(a) at 25° C. of 1 to 7, in particular of 2 to 6.5, more particularlyof 3 to 6.5.

The pH of the separately packaged volume containing the EL-POXpreferably lies in the range of 2-7, more preferably of 3-6, mostpreferably of 4-5.

The separately packaged volume containing the nucleophilic agentpreferably contains an alkalinizing agent that shifts themicro-environmental pH of the nucleophilic agent containing volume tomore alkaline conditions in the presence of water or bodily fluids.

Examples of suitable alkalinizing agents include ammonia solution,ammonium carbonate, alkali metal salts including alkali metal carbonates(for instance potassium carbonate and sodium carbonate), potassiumhydroxide, sodium hydroxide and sodium borate (borax), or tertiaryamines such as triethylamine, triethanolamine or other amines likediethanolamine and monoethanolamine.

Preferably, the alkalinizing agent has a pK_(a) at 25° C. of 8-14, inparticular of 8.5-11, more particularly of 9 to 11.

The pH of the separately packaged volume containing the nucleophilicagent preferably lies in the range of 7-12, more preferably of 8-11,most preferably of 9-10.

The separately packaged volumes EL-POX and cross-linking agent areadvantageously contained within a dispensing means from which bothvolumes can be dispensed simultaneously. Thus, the two cross-linkingagents may be delivered simultaneously and a cross-linked polymer willform in situ. By simultaneously dispensing the two reactants anddelivering them at the site where they cross-linking should occur,pre-mixing can be avoided and premature cross-linking is effectivelyprevented. Examples of suitable dispensing means include spraydispensers, syringes, and dual syringes. Syringes suitably comprise astatic mixer that ensures that the two reactants are mixed together whensimultaneously expelled from said syringe.

In accordance with a preferred embodiment at least one of the separatelypackaged volumes contains a visualization agent to enhance thevisibility. The visualization agent (e.g. a colourant) reflects or emitslight at a wavelength detectable to a human eye. Because of theinclusion of the visualization agent it is easy for users to accuratelyapply the reactive mixture. Examples of suitable colourants include FD&Cand D&C colorants, such as FD&C Violet No. 2, FD&C Blue No. 1 , D&CGreen No. 6, D&C Green No. 5, D&C Violet No. 2; and natural colorantssuch as beetroot red, canthaxanthin, chlorophyll, eosin, saffron,carmine, indocyanine green, or colored dyes normally found in syntheticsurgical sutures. Similarly, dyes such as fluoroscein and methylene bluecan be used. The visualization agent may or may not become chemicallybound to the hydrogel.

In an alternative embodiment, the kit comprises a first biocompatiblethin film containing the EL-POX and a second biocompatible thin filmcontaining the nucleophilic cross-linking agent. The two films maysuitably be applied after combining the two films on top of each other.Films may suitably have been perforated to increase their specificsurface area.

The biocompatible thin film containing the nucleophilic cross-linkingagent preferably comprises an alkalinizing agent as defined hereinbefore.

In accordance with another alternative embodiment, the kit comprisesparticles having a weight averaged mean diameter of 0.01-1000 μm, saidparticles including particles containing EL-POX and particles containingthe nucleophilic cross-linking agent. The EL-POX and the nucleophiliccross-linking agent may be contained in the same particles or they maybe contained in different particles within the same powder.

The aforementioned powder may suitably be used as a haemostatic powderthat can be stored under ambient conditions.

Besides the EL-POX and/or the nucleophilic cross-linking agent, theparticles comprised in the haemostatic powder may suitably contain acarrier, preferably a water-soluble carrier. Examples of carriers thatmay suitably be employed include monosaccharides; di- andoligosaccharides, such as lactose, mannitol, trehalose, erythritol,xylitol, sorbitol, maltitol, isomalt, maltodextrin, cellobiose, glucose,fructose, maltulose, lactulose, maltose, gentobiose, isomaltose,lactitol, palatinitol, dulcitol, ribitol, sucrose, raffinose,gentianose, planteose, verbascose, stachyose, melezitose, inositol; andpolysaccharides, such as dextran, starch (amylose, amylopectin),glycogen, cellulose, chitin, alginates, callose, chrysolaminarin, xylan,arabinoxylan, mannan, fucoidan and galactomannan; and combinations ofthese carbohydrates. Preferably, the carrier employed has a glasstransition temperature of at least 0° C., more preferably of at least25° C.

Typically, the haemostatic powder contains 25-75 wt. % of the EL-POX,25-75 wt. % of the nucleophilic cross-linking agent and 0-50 wt. % ofcarrier.

Yet another aspect of the invention relates to a tissue adhesive medicalproduct comprising at least 1%, preferably at least 50% and mostpreferably at least 90% by weight of dry matter of EL-POX, said EL-POXcomprising at least 2 electrophilic groups, including at least onependant electrophilic group, said reactive groups being selected fromcarboxylic acid esters, sulfonate esters, phosphonate esters,pentafluorophenyl esters, p-nitrophenyl esters, p-nitrothiophenylesters, acid halide groups, anhydrides, ketones, aldehydes, isocyanato,thioisocyanato, isocyano, epoxides, activated hydroxyl groups, olefins,glycidyl ethers, carboxyl, succinimidyl esters, succinimidyl carbonates,succinimidyl carbamates, sulfosuccinimidyl esters, sulfosuccinimidylcarbonates, maleimido, ethenesulfonyl, imido ester, aceto acetate, haloacetal, orthopyridyl disulfide, dihydroxyphenyl derivatives, vinylacrylate, acrylamide, iodoacetamide and combinations thereof. Theelectrophilic groups in the EL-POX may suitably be selected fromcarboxylic acid esters, acid chloride groups, anhydrides, ketones,aldehydes, isocyanato, thioisocyanato, epoxides, activated hydroxylgroups, olefins, carboxyl, succinimidyl ester, succinimidyl carbonate,succinimidyl carbamates, sulfosuccinimidyl ester, sulfosuccinimidylcarbonate, maleimido, ethenesulfonyl and combinations thereof.

Preferably, the electrophilic groups in the EL-POX are selected fromaldehydes, imido esters, isocyanato, thioisocyanato, succinimidylderivatives, sulfosuccinimidyl derivatives, dihydroxyphenyl derivatives,halo acetals, orthopyridyl disulfide, maleimides, vinyl sulfone andcombinations thereof.

Even more preferably, the electrophilic groups contained in the EL-POXare selected from aldehydes, succinimidyl esters, succinimidylcarbonates, succinimidyl carbamates, imido esters, dihydroxyphenylderivatives, halo acetals, maleimides and combinations thereof.

Most preferably, the electrophilic groups are selected from the group ofN-hydroxysuccinimide esters, aldehydes, maleimides, dihydroxyphenylderivatives and combinations thereof

In accordance with this particular embodiment of the invention it ispreferred that the EL-POX contained in the tissue adhesive medicalproduct has not, or only partially, been cross-linked. Most preferably,the EL-POX contained in the tissue adhesive medical product has not beencross-linked.

The tissue adhesive product of the present invention may comprise anucleophilic cross-linking agent as defined herein before. It ispreferred that the number of reactive nucleophilic groups provided bysaid cross-linking agent is significantly lower than the number ofreactive electrophilic groups contained in the EL-POX. Thus, it isensured that sufficient numbers of electrophilic groups will remainafter the cross-linking reaction between EL-POX and the nucleophiliccross-linking agent to provide adhesion between the cross-linked polymerand e.g tissue. This advantageous embodiment may be realized, forinstance, by plasticizing EL-POX and the nucleophilic cross-linkingagent into a single film or tape. Before application as a tissue tape,the latter tape may be combined with another water soluble filmcontaining an activator, or a fluid or a spreadable compositioncontaining an activator.

In accordance with a particularly preferred embodiment, the tissueadhesive medical product comprises at least 1%, preferably at least 50%and most preferably at least 75% by weight of dry matter of EL-POX andat least 1%, preferably at least 50% and most preferably at least 75% byweight of dry matter of NU-POX. Advantageously, EL-POX and NU-POXtogether represent at least 75 wt. %, more preferably at least 95 wt. %of the dry matter contained in the tissue adhesive product.

The combined use of EL-POX and NU-POX in the tissue adhesive medicalproduct offers the advantage that the product can be produced in theform of an amorphous film, tape or powder, e.g. by melt extrusion.

An advantage associated with employing a combination of EL-POX andNU-POX in non-crosslinked form lies in the fact that it enables thepreparation of very flexible films or tapes that area easily appliedduring surgery. Furthermore, the EL-POX and NU-POX will rapidly react toform a cross-linked polymer as soon as water is absorbed by the tissueadhesive medical product (e.g. film or tape), thereby providingexcellent sealing properties.

In accordance with another embodiment, the tissue adhesive medicalproduct comprises no nucleophilic cross linking agent. The EL-POX maysuitably be embedded in a matrix of other (non-reactive) film formingpolymers, including polyoxazolines such as polyethyloxazoline,polymethyloxazoline or polypropyloxazoline. Preferably, the adhesivemedical product in accordance with this embodiment comprises 10-95%,more preferably 30-80% and most preferably 50-70% EL-POX by weight ofdry matter. The other film forming polymers, preferably represent 5-90%,more preferably 20-70% and most preferably 30-50% of the dry mattercontained in the adhesive medical product.

The EL-POX comprised in the tissue adhesive medical product preferablycontains at least 2 electrophilic groups per 100 monomers, morepreferably at least 5 electrophilic groups per 100 monomers, even morepreferably 10-80 electrophilic groups per 100 monomers and mostpreferably 10-50 electrophilic groups per 100 monomers.

Examples of tissue adhesive medical products that are encompassed by thepresent invention include adhesive tissue tape, tissue sealant,haemostatic porous material and implants.

Medical products in the form of a tape or film may suitably comprise apolymer matrix comprising the EL-POX and a water-insoluble backingmembrane. The use of a backing membrane or film ensures that the EL-POXdoes not induce unwanted adhesions as the covalent attachment tosurrounding tissue will be prevented. The backing membrane may be madeof any suitable biocompatible polymer. Preferably, the backing membraneis made of a biocompatible polymer containing primary amines as thesewill covalently link to the EL-POX.

The crosslinked polymer according to the present invention, the EL-POXas well as the NU-POX may advantageously contain an antimicrobial agentcovalently bound to one of the oxazoline unit comprised therein. Morepreferably, the crosslinked polymer or EL-POX contains an antimicrobialagent that is covalently bound to oxazoline units through an amide orimide group. Examples of antimicrobial agents include aminophenols,aminocresol, amino resorcinol and aminonaphtol. The antimicrobial agentmay suitably be bound to the POX-polymer via a spacer group, such as analkylene, oxyalkylane or silicone. By covalently binding anantimicrobial agent to the POX-polymer it can be ensured that theantimicrobial agent is slowly released during biodegradation of thepolymer.

A further embodiment of the present invention provides a compositioncomprising an adhesive film containing:

-   -   a polymeric material selected from EL-POX, the EL-POX derived        biocompatible, covalently cross-linked polymer according to the        present invention and combinations thereof; and    -   one or more dihydroxyphenylalanine (DHP) derivatives.

The DHP derivatives are preferably selected from3,4-dihydroxyphenylalanine (DOPA), dopamine, 3,4-dihydroxyhydroccinamicacid (DOHA), catechol, and combinations thereof.

Typically, the adhesive film contains at least 1%, more preferably atleast 25%, most preferably 50-100% of the polymeric material by weightof dry matter.

The DHP content of the adhesive film typically lies in the range of10-95%, more preferably 20-80% and most preferably 40-60% by weight ofdry matter.

The water content of the adhesive film preferably does not exceed 5wt.%, more preferably it does not exceed 1 wt. %.

The composition may suitably further comprise one or more additivecomponents selected from fillers, oxidants, crosslinkers, microgels,additional polymers, drugs and other therapeutic agents.

The adhesive film may consist of a single layer or it may be composed ofa plurality of layers.

The reaction between tissue-reactive electrophilic groups and functionalgroups on the surface of the tissue may vary with pH. It may thereforebe preferable to buffer the tissue surface immediately prior toapplication or, more preferably, to include a buffer in the productcontaining covalently cross-linked polymer with reactive elecrophilicgroups and/or EL-POX. In the case such product is, for instance, anadhesive film or tape, it may be advantageous to include a buffer in thetissue-contacting layer of the film or tape. The adhesion of suchproducts may be improved by buffering the tissue surface of the productwith a buffer having a pH in the range of 9.0-12.0, especially of9.5-11.5.

The medical products according to the present invention mayadvantageously be used in a variety of surgical applications. Examplesof these surgical applications are summarized below.

Neurosurgery Repair of dural defects; repair of central nervous systemtissue; spinal cord repair; nerve grafting; intervertebral disc surgeryand cerebrospinal fluid leaks (CSF leaks). Ophthalmic surgery Clearcorneal cataract surgery; laser in situ keratomileusis (LASIK) surgery;corneal ulcer treatment; corneal transplantation; conjunctival repair;retinal attachment; punctal plugging for treatment of dry eyes;oculoplastics and blepharoplasty (eyelid lifts); vitrectomy closure andattachment of extraocular muscles. Ear, nose and throat Control ofepistaxis (nosebleeds); repair of vocal cord defects; surgerytympanoplasty for repair of perforated eardrum; myringotomy (eardrumincision for drainage) with tube insertion; sinus surgery; nasalreconstructive surgery; tonsillectomy surgery and adenoidectomy surgery.Head and neck Salivary gland removal; lymph node dissection andtreatment surgery of chylous leakage after neck dissection.Interventional Therapeutic embolization and femoral artery closureduring radiology interventional procedures. Vascular surgeryArteriovenous fistula repair, aortic aneurysm repair and vascularanastomosis. Cardiovascular Cardiac valve repair; repair of ventricularwall rupture; surgery coronary artery anastomosis during bypass surgery;pacemaker and lead placement; aortic anastomosis and treatment of aorticdissection. Thoracic surgery Lung lobectomy; lung biopsy andpneumothorax treatment. Gastrointestinal gastrointestinal anastomosis;peptic ulcer treatment; treatment surgery of esophageal rupture;gallbladder or bile duct anastomosis; gastric bypass surgery;appendectomy; cholecystectomy (gallbladder removal); pancreatic surgery;gastrointestinal fistula repair; sealing of peritoneal dialysis catheterleakage, treatment of abdominal hernias and prevention of intra-abdominal adhesions Colorectal surgery Colonic anastomosis; rectalfistula repair; treatment of diverticular bleeding; hernia patchplacement and hemorrhoidectomy. Liver surgery Liver resection and livertransplantation. Gynecologic surgery Hysterectomy; myomectomy foruterine fibroid removal; fallopian tube anastomosis; vaginal fistularepair; cervical surgery; ovarian cyst removal; breast biopsy;mastectomy and lumpectomy and management of preterm premature rupture ofmembranes. Urologic surgery Nephrectomy, kidney transplantation;urethral fistula repair; urethral anastomosis; repair for stress urinaryincontinence; bladder closure; radical prostatectomy and vasectomyreversal surgery. Pediatric surgery Congenital cleft lip repairOrthopedic surgery Hip replacement surgery; knee replacement surgery;tendon reattachment; cartilage repair; intervertebral disc repair,fracture repair and bone grafting Plastic and Face lift surgery; closureof skin incisions; soft tissue reconstructive augmentation surgeryTrauma surgery Closure of splenic lacerations and other solid organs;closure of skin lacerations; bleeding control during burn debridementand skin grafting for burn victims.

Polymeric coated gas bubbles acting as ultrasound contrast agents cansuitably be embedded in the kits or the medical products describedherein before. Polymeric coated gas bubbles derive their contrastproperties from the large acoustic impedance mismatch between blood andthe gas contained therein. Examples of polymers that can be used to coatthese gas bubbles include polylactide, polyglycolide, polycaprolactone,copolymers of polylactide and polyglycolide, copolymers of lactide andlactone, polysaccharide, polyanhydride, polystyrene,polyalkylcyanoacrylate, polyamide, polyphosphazene,poly(methylmethacrylate), polyurethane, copolymers of methacrylic acidand acrylic acid, copolymers of hydroxyethylmethacrylate andmethylmethacrylate, polyesters, such as polycarbonates, and protein.Preferred polymers are those which are biocompatible and/orbiodegradable. In a preferred embodiment the polymer is polylacticco-glycolic acid (PLGA).

Alternatively, the EL-POX, NU-POX or the cross linked polymer may beproduced in such way that gas bubbles are entrapped therein. Preferably,a gas is formed in-situ by reaction of a pH modifier (formulated in theEL-POX phase) with a carbon dioxide releasing base (formulated in theNU-POX phase). Even more preferably, the pH modifier and the carbondioxide releasing base are formulated in the combined NU-POX/EL-POXphase in which the pH modifier is triggered to lower the pH upon contactwith water or bodily fluids

The invention is further illustrated by means of the followingnon-limiting examples.

EXAMPLES Example 1

Amphiphilic copolymers of EL-POX containing ethyl and NHS groups in thealkyl side chain were synthesized by cationic polymerization of2-ethyl-2-oxazoline (ETOX) and 2-(3-butenyl)-2-oxazoline (BUTOX) toyield a poly[2-(ethyl/3-butenyl)]-2-oxazoline copolymer.

This poly[2-(ethyl/3-butenyl)]-2-oxazoline copolymer was functionalizedin a two-step approach by first thiol-ene coupling of mercaptopropionicacid followed by activation with NHS to synthesize NHS-side chainactivated poly[2-(ethyl/3-butenyl)]-2-oxazoline copolymer. The initialETOX/BUTOX molar ratio was 90/10 and the percentage of reactive groupsper poly[2-(ethyl/3-butenyl)]-2-oxazoline copolymer, was therefore 10%,as explained before.

An amount of 84.3 mg of this NHS-side chain activatedpoly[2-(ethyl/3-butenyl)]-2-oxazoline copolymer was dissolved in 150 μLethanol (absolute) and mixed with 150 μL of a mixture of water, ethanoland triethylamine (1:1:1 v/v/v) containing 9.5 mg trilysine triacetate.The combined fluid mixture (about 300 μL containing 28wt % polymer and aNHS-amine ratio of 1:1.25) turned into a cross linked network (gel)within 30 to 45 seconds.

Example 2

NHS-side chain activated poly[2-(ethyl/3-butenyl)]-2-oxazoline copolymerwas synthesized as described in example 1.

An amount of 300 mg of this NHS-side chain activatedpoly[2-(ethyl/3-butenyl)]-2-oxazoline copolymer was dissolved in 750 μLethanol (absolute) and 150 μL of trilysine triacetate (10 mg) inethanol/water 50/50 v/v, containing about 10 μL triethyl amine wasadded.

The burst pressure of this formulation was tested in the followingmanner. Briefly, fresh bovine dura was dissected into suitably sizedsections of tissue. A 3 mm circular incision was cut into the dura,which was then secured to a burette in such a manner that simulated bodyfluid could be forced out under measurable pressure via the circularincision.

The formulation containing the active POX copolymer was dried to anamorphous tape by means of vacuum at room temperature and put onto thesecured dura to form a seal. After a few minutes, the pressure of thesimulated body fluid was increased to the point that the seal burst(burst pressure). The burst pressure was found to be 75 mmHg. TheNHS:amine ratio in this film is 2.7:1.

Comparative Example A

The experiment described in Example 2 was repeated (using the samedura), except that this time the film consisted of non-side chainactivated poly-ethyl-oxazoline film. This time the burst pressure wasfound to be about 15 mmHg.

These results show that the incorporation of reactive cross-linkinggroups in the poly[2-(ethyl/3-butenyl)]-2-oxazoline copolymer, such asNHS, significantly increases the adhesion strength of the tape to thecollagen of bovine dura.

Example 3

Amphiphilic copolymers of EL-POX containing ethyl and NHS groups in thealkyl side chain were synthesized by controlled acidic hydrolysis ofaquazol 50 (poly(2-ethyl-2-oxazoline (PEtOx), Mw 50,000) to yield apoly[2-ethyl-2-oxazoline/ethylene imine] copolymer (PEtOx-PEI) in which6% of the initial 2-ethyl-oxazoline units were hydrolyzed.

This poly[2-ethyl-2-oxazoline/ethylene imine] was functionalized in athree-step approach by coupling of methyl succinyl chloride in thepresence of triethylamine, followed by the hydrolysis of the methylester with lithium hydroxide followed by activation of the obtainingcarboxylic acid moieties with NHS in the presence of EDC to synthesizeNHS-side chain activated poly(2-ethyl /NHS-ester-ethyl)-2-oxazolinecopolymer. The final functionalisation with NHS of the copolymer wasdetermined by UV-vis spectroscopy to be 4.4%.

An amount of 15 mg of this NHS-side chain activatedpoly[2-(ethyl/NHS-ester-ethyl)]-2-oxazoline copolymer was dissolved in217 μL ethanol (absolute) and mixed with 16 μL of a mixture of water,ethanol and triethylamine (1:1:1 v/v/v) containing 1.0 mg trilysine. Thecombined fluid mixture (about 230 μL containing ˜6.5 wt % polymer withan NHS-amine ratio of 1.01:1.00) turned into a cross linked network(gel) within 60 seconds at room temperature.

Example 4

NHS-side chain activated poly[2-(ethyl/NHS-ester-ethyl)]-2-oxazolinecopolymer was synthesized as described in example 3.

An amount of 410 mg of this NHS-side chain activated poly[2-(ethyl/NHS-ester-ethyl)]-2-oxazoline copolymer was dissolved in 8.25 mL ethanol(absolute) and a solution containing trilysine (5 mg) in 23 μL ethanol,10 μL water and 10 μL triethyl amine was added. The NHS:amine ratio inthis film is 5.5:1.

The burst pressure of this formulation was tested as described inexample 2. Instead of fresh bovine dura, bovine peritoneum was used.

The formulation containing the active POX copolymer was dried to anamorphous tape at 40 degrees Celsius and put onto the secured peritoneumto form a seal. After a few minutes, the pressure of the simulated bodyfluid was increased to the point that the seal burst (burst pressure).The burst pressure was found to be 11 mmHg.

Comparative Example B

An amorphous tape, made in the same way as describe in Example 4, withNHS:amine ratio equals 1:1, was put onto the secured peritoneum to forma seal. After a few minutes, the pressure of simulated body fluid wasincreased to the point that the seal burst (burst pressure). The burstpressure was found to be 3.7 mm Hg, indicating the importance of havingan excess of electrophilic groups to ensure tissue adhesive properties.

Comparative Example C

The ‘burst’ experiment described in Example 4 was repeated with a film(tape) consisting of non-side chain activated aquazol 50(poly(2-ethyl-2-oxazoline) (PEtOx), Mw 50,000) film. For this experimentthe burst pressure was found to be about 2.6 mmHg. Upon contact withwater this film starts to dissolve.

This result shows that incorporation of reactive cross-linking groups inthe poly[2-(ethyl/NHS-ester-ethyl)]-2-oxazoline copolymer, such as NHS,significantly increases the adhesion strength of the tape to thecollagen of bovine peritoneum.

Example 5

An amorphous tape, made in the same way as described in example 4, butonly using the poly[2-(ethyl/NHS-ester-ethyl)]-2-oxazoline copolymer,was put onto the secured peritoneum to form a seal. After a few minutes,the pressure of simulated body fluid was increased to the point that theseal burst (burst pressure). The burst pressure was found to be 3.7 mmHg. Upon contact with water this film also starts to dissolve.

The adhesion of this tape was excellent. Due to the lack of anucleophilic cross-linker cohesion was the limiting factor, leading to amoderately low burst pressure (the film was ruptured). Cohesion of thisadhesive tape can be improved e.g. by combining the copolymer with ahydrophobic support.

Example 6

NHS-side chain activated poly[2-(ethyl/NHS-ester-ethyl)]-2-oxazolinecopolymer was synthesized as described in example 3.

Amine-side chain activated poly[2-(ethyl/NHS-ester-ethyl)]-2-oxazolinecopolymer (NU-POX) was synthesized by controlled acidic hydrolysis ofaquazol 50 (poly(2-ethyl-2-oxazoline (PEtOx), Mw 50,000) to yield apoly[2-ethyl-2-oxazoline/ethylene imine] copolymer (PEtOx-PEI) in which12% of the initial 2-ethyl-oxazoline units was hydrolyzed.

This poly[2-ethyl-2-oxazoline/ethylene imine] was functionalized in atwo-step approach by first coupling of methyl succinyl chloride in thepresence of triethylamine, followed by the reaction of the formed methylester side chain with ethylenediamine to yield an amine-side chainactivated poly(2-ethyl /amino-ethyl-amide-ethyl)-2-oxazoline copolymer.

A solution containing this NU-POX (3.4 mg) in 250 μL borate buffer (0.1M, pH 8.5) was added to a solution of NHS-side chain activatedpoly[2-(ethyl/NHS-ester-ethyl)]-2-oxazoline copolymer (10.0 mg)dissolved in 100 μL H₂O. The combined fluid mixture (about 350 μLcontaining ˜4 wt % polymer with an NHS-amine ratio of 1.01:1.00) turnedinto a cross linked network (gel) within 20 seconds at room temperature.

Example 7

NHS-side chain activated poly[2-(ethyl/NHS-ester-ethyl)]-2-oxazolinecopolymer was synthesized as described in example 3.

Amine-side chain functionalizedpoly(2-ethyl/amino-ethyl-amide-ethyl)-2-oxazoline copolymer (NU-POX) wassynthesized as described in example 6.

An amount of 266 mg of this NHS-side chain activated poly[2-(ethyl/NHS-ester-ethyl)]-2-oxazoline copolymer was dissolved in 1.33 mL ethanol(absolute) and a solution containing NU-POX (18.4 mg) in 822 μL ethanol(absolute) and 27 μL triethylamine was added. The solution containingthe active POX copolymers was dried to an amorphous tape at 40 degreesCelsius and put onto the secured peritoneum to form a seal. The burstpressure of this formulation was determined as described in example 4.The NHS:amine ratio in this film is 5.5:1.

After a few minutes, the pressure of the simulated body fluid wasincreased to the point that the seal burst (burst pressure). The burstpressure was found to be 23 mmHg, which is significantly higher than the2.6 mmHg burst pressure of the reference experiment described in Example5.

These results again show that the incorporation of reactivecross-linking groups in the poly[2-(ethyl/NHS-ester-ethyl)]-2-oxazolinecopolymer, such as NHS, significantly increases the adhesive strength ofthe tape to the collagen of bovine peritoneum. The adhesion of this tapewas excellent. Due to the nucleophilic cross linker (NU-POX), thecohesion was good, leading to a relatively high burst pressure. When theburst occurred, the film was not ruptured, meaning that in this caseloss of adhesion rather than loss of cohesion was the limiting factor.

Example 8

Amine-side chain functionalizedpoly(2-ethyl/amino-ethyl-amide-ethyl)-2-oxazoline copolymer wassynthesized as described in example 6. This NU-POX was turned into anEL-POX by reacting the amine with maleic anhydride to yieldmaleimide-side chain activated poly(2-ethyl/maleimide-ethyl-amide-ethyl)-2-oxazoline copolymer.

A thiol derivatized polyethylene glycol (substituted four-arm PEG)polymer was synthesized from an amine derivatized polyethylene glycol(PEG) polymer component consisting of pentaerythritol polyethyleneglycol ether tetra-amine. The amine groups were coupled toS-acetylthioglycolic acid N-hydroxysuccinimide ester. After removal ofthe acetyl protecting group of the thiol, the thiol substituted four-armPEG was obtained.

An amount of 17.5 mg of the maleimide-side chain activatedpoly[2-(ethyl/maleimide-ethyl-amide-ethyl]-2-oxazoline copolymer wasdissolved in 100 μL ethanol (absolute) and mixed with 102 μL of amixture of water and triethylamine (100:2 v/v) containing 32 mg of thiolsubstituted four-arm PEG thiol. The combined fluid mixture (about 200 μLcontaining 25 wt % polymer with a maleimide-amine ratio of 3:2) turnedinto a cross linked network (gel) within 20 seconds at room temperature.

The invention claimed is:
 1. A tissue-adhesive biocompatible, medicalproduct comprising at least 1% by weight of dry matter of abiocompatible, covalently cross-linked, electrophilically activatedpolyoxazoline (EL-POX) having side chains containing an electrophilicgroup, wherein of the total number of reacted and un-reactedelectrophilic groups in the cross-linked EL-POX, 10-95% comprise freeelectrophilic groups, and wherein the tissue-adhesive biocompatible,medical product exhibits stickiness.
 2. The biocompatible, medicalproduct according to claim 1, wherein at least one of the freeelectrophilic groups is a pendant electrophilic group.
 3. Thebiocompatible, medical product according to claim 2, comprising 3 to 50pendant electrophilic groups per 100 monomers.
 4. The biocompatiblemedical product according to claim 1, wherein the cross-linked polymeris in an amorphous state.
 5. The biocompatible, medical productaccording to claim 1, selected from implants, tissue sealants, adhesivetissue tape, adhesive tissue film, suture material, polymer coatedstents and haemostatic materials.
 6. The biocompatible, medical productaccording to claim 1, comprising less than 5% water by weight of thecross-linked polymer.
 7. The biocompatible, medical product according toclaim 1, wherein the EL-POX has a molecular weight of at least 5000g/mol.
 8. The biocompatible, medical product according to claim 7,wherein the EL-POX has a molecular weight of at least 10000 g/mol. 9.The biocompatible, medical product according to claim 8, wherein theEL-POX has a molecular weight of at least 30000 g/mol.
 10. The medicalproduct according to claim 1, wherein the electrophilic groups areselected from the group consisting of carboxylic acid esters, sulfonateesters, phosphonate esters, pentafluorophenyl esters, p-nitrophenylesters, p-nitrothiophenyl esters, acid halide groups, anhydrides,ketones, aldehydes, isocyanato, thioisocyanato, isocyano, epoxides,activated hydroxyl groups, olefins, glycidyl ethers, carboxyl,succinimidyl esters, succinimidyl carbonates, succinimidyl carbamates,sulfosuccinimidyl esters, sulfosuccinimidyl carbonates, maleimido,ethenesulfonyl, imido ester, aceto acetate, halo acetal, orthopyridyldisulfide, dihydroxyphenyl groups, vinyl acrylate, acrylamide,iodoacetamide and combinations thereof.
 11. The medical productaccording to claim 10, wherein the electrophilic groups are selectedfrom the group consisting of carboxylic acid esters, acid chloridegroups, anhydrides, ketones, aldehydes, isocyanato, thioisocyanato,epoxides, activated hydroxyl groups, olefins, carboxyl, succinimidylester, succinimidyl carbonate, succinimidyl carbamates,sulfosuccinimidyl ester, sulfosuccinimidyl carbonate, maleimido,ethenesulfonyl and combinations thereof.
 12. The biocompatible, medicalproduct according to claim 11, wherein the electrophilic groups areselected from the group consisting of aldehydes, isocyanato,thioisocyanato, succinimidyl ester, sulfosuccinimidyl ester, maleimidoand combinations thereof.
 13. The biocompatible, medical productaccording to claim 12, wherein the electrophilic groups are selectedfrom the group consisting of isocyanato, thioisocyanato, succinimidylester, sulfosuccinimidyl ester, maleimido and combinations thereof. 14.The biocompatible, medical product according to claim 1, wherein theelectrophilic groups are selected from the group consisting ofN-hydroxysuccinimide esters, aldehydes, maleimides, dihydroxyphenylgroups and combinations thereof.
 15. The biocompatible, medical productaccording to claim 1, comprising 10% by weight of dry matter of theEL-POX.
 16. The biocompatible, medical product according to claim 15,comprising 25% by weight of dry matter of the EL-POX.
 17. Thebiocompatible, medical product according to claim 1, wherein of thetotal number of reacted and non-reacted electrophilic groups in thecross-linked EL-POX, 40-95% comprise free electrophilic groups.